Project Summary The stochastic and monoallelic expression of one out of a thousand olfactory receptor (OR) genes in mammals is a complex process governed by the spatial compartmentalization of active and silent OR alleles in olfactory sensory neurons (OSNs). During OSN differentiation OR loci from multiple chromosomes converge into distinct, OSN-specific nuclear foci characterized by the hallmarks of constitutive heterochromatin. Absent from these unusual nuclear bodies is the OR allele that is transcriptionally active in each OSN, which typically resides on euchromatic nuclear compartments and is surrounded by numerous enhancer elements recruited from several chromosomes. This intricate network of interchromosomal interactions is responsible for both the robust transcription of the chosen OR allele and the complete silencing of the repressed ones. The extraordinary number of OR family members and the unprecedented extent of long-range genomic interactions that culminate to the remarkable organization of the OR nucleome, make the olfactory system ideal for they study of the molecular principles that organize the mammalian nuclear architecture in vivo. For a comprehensive interrogation of the OR nucleome, we assembled a multidisciplinary team seeking to combine novel genetic manipulations with a one of a kind imaging system, a state of the art proteomics facility, and innovative genomic analyses. With CRISPR, phiC31 integrase and in utero DNA electroporation we will tag OR loci and enhancers, making the OR subgenome accessible by three novel experimental strategies: High resolution imaging by correlated soft X-ray tomography and cryo- SIM; biochemical purification by sequence specific tagging with Halo and APEX followed by sophisticated mass spectrometry; and genomic analysis of long range interactions occurring during OSN differentiation using two different DNA modifying enzymes and single molecule real time sequencing. This ambitious experimental project not only will reveal molecular mechanisms that govern the nuclear organization of OR genes but also generally applicable principles and powerful technologies for the study of the mammalian nucleome in vivo.